Cobalamins (Cbl, vitamin B12 derivatives) are essential cofactors in mammals. However, they also have clinical effects that cannot be fully explained solely by their cofactor role. For example, B12 has been used extensively by physicians over several decades to treat inflammatory disorders including chronic fatigue syndrome, rheumatoid arthritis, asthma, neurological diseases and eczema, with little understanding as to why B12 supplementation is beneficial. These diseases are associated with "oxidative and nitrosative stress" - elevated levels of Reactive Oxygen and Nitrogen Species (ROS/RNS), such as nitric oxide, superoxide, the hydroxyl radical, hydrogen peroxide and peroxynitrite. ROS/RNS react with biomolecules including proteins, DNA and lipids, frequently causing irreversible tissue and organ damage. We hypothesize that pharmacological doses of Cbl protect against ROS/RNS-initiated cell damage. We further hypothesize that an important mechanism for this protection is the direct scavenging of ROS/RNS by a major intracellular Cbl form, cob(II)alamin. The overall goal of this study is to determine whether Cbl is a broad spectrum antioxidant, capable of directly scavenging multiple ROS/RNS. In Specific Aim 1 the reactions between cob(II)alamin, Cbl(II), and four ROS/RNS species - hydrogen peroxide, superoxide, the hydroxyl radical and nitrogen dioxide - will be studied. Kinetic and product studies will allow rates and reaction mechanisms to be elucidated. This will provide invaluable information about the potential biological relevance of these reactions. In Specific Aim 2 CNCbl's ability to scavenge excess ROS and RNS in primary human aortic endothelial cells and prevent ROS/RNS-initiated cell death will be determined. CNCbl is the common Cbl form in vitamin supplements and is reduced to Cbl(II) upon entering cells. The vascular endothelium plays an important role in regulating blood flow, in inhibiting platelet aggregation and in the immune and inflammatory response. Vascular endothelial dysfunction is associated with oxidative/nitrosative stress. The ability of CNCbl to scavenge individual ROS/RNS (hydrogen peroxide, superoxide, the hydroxyl radical, and the nitrogen dioxide source peroxynitrite) will be determined using intracellular fluorescent or chemiluminescent probes and/or downstream assays specific for each ROS/RNS. The protective effects of CNCbl against cell death induced by each ROS/RNS will also be investigated. The current proposal challenges the widely-held belief that the sole biological role of Cbl is its coenzyme role in B12-dependent enzyme reactions. It also challenges the common belief that free (non-protein bound) Cbl has no biological role, despite the fact that ~50% free Cbl is achieved in cells upon supplementation, and that Cbl(II) is a radical species capable of reacting rapidly with ROS/RNS. The long-term goal is to identify the mechanism(s) by which Cbl protects against cellular oxidative/nitrosative stress, in order to assess the benefits of Cbl supplementation in the treatment of chronic inflammatory diseases. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE This study explores a novel mechanism by which vitamin B12 can provide protection against inflammatory and age-related diseases. Given a rapidly aging population, the study of the therapeutic potential of vitamin B12 is highly relevant since B12 deficiency is increasingly common, especially amongst the elderly.